Within the scope of microscopic fluorescence examinations of specimens, the specimens are usually illuminated directly with an illumination light bundle in order to optically excite the specimen in the illuminated region and to subsequently detect the fluorescent light that is being emitted by the specimen. In scanning microscopy, the focus of an illumination light bundle is directed over or through the specimen in a meandering pattern, generally using a controllable beam deflector that can comprise, for example, one or more tilting mirrors, and in this manner, the specimen is scanned one point at a time.
As an alternative to direct specimen illumination, it is also possible to illuminate the specimen evanescently. Here, the excitation light is totally reflected on a boundary surface to the specimen, whereby the specimen is excited by the evanescent electromagnetic field that diminishes with the penetration depth. The term TIRFM (total internal reflection fluorescence microscopy) has become established for this type of specimen examination.
German patent application DE 103 44 410 A1 discloses a scanning microscope with evanescent specimen illumination. The scanning microscope comprises a light source whose light is coupled into a cover glass so that the light can propagate over the surface of a cover glass through total internal reflection and can illuminate a large surface area of a specimen that is arranged on the cover glass. Moreover, the scanning microscope has a point detector that receives detection light that is emitted by a scanned point of the specimen, and it also has a beam deflector arranged in the beam path of the detection light in order to shift the position of the scanned point in the specimen. However, this device has the drawback that the coupling of the illumination light into the cover glass is not very efficient, as a result of which only a reduced quantity of light is available to evanescently illuminate the specimen.
Moreover, when it comes to the evanescent illumination of specimens, the methods and devices known from the state of the art often have the drawback that the precise location of the specimen illumination cannot be set flexibly and sufficiently precisely.
Moreover, the methods and devices known from the state of the art entail the disadvantage that, prior to a microscopic examination under evanescent illumination, the specimens have to be laboriously arranged and prepared in special specimen chambers or between cover glasses before the specimen chamber or the arrangements of cover glasses between which the specimen is mechanically clamped can be laid onto the object stage of a microscope. It is often the case that precisely the edge regions of the specimen that are located at the boundary surface where the total reflection is supposed to take place are damaged by the continuous pressure stress, so that the subsequent microscopic examination is at the minimum negatively influenced or even rendered completely impossible.